Journal of the Pancreas Open Access

Keywords

Angiotensin II; Insulin Resistance; NADPH Oxidase; Oxidative Stress; Reactive Oxygen Species; Renin

Abbreviations

Alpha-SMA: alpha smooth muscle actin; T2DM: type 2 diabetes mellitus; RAAS: renin-angiotensin-aldosterone system; TEM: transmission electron microscopy

INTRODUCTION

Type 2 diabetes mellitus (T2DM) has reached pandemic proportions and current predictions are that this trend will increase [1, 2]. Thus, a better understanding of islet reninangiotensin- aldosterone system (RAAS) mechanisms may help in ascertaining how islet structure and function are involved in the early changes associated with the overexpression of islet tissue RAAS. Therefore, we have chosen to study the transgenic (mRen2)27 control rat model of hypertension and insulin resistance (due to transfection of the mouse renin gene), which is known to result in the overproduction of local tissue angiotensin II in the heart, kidney, skeletal muscle and presumably the islet [3, 4, 5, 6, 7, 8].

There have been a number of publications regarding the role of an active RAAS in the islet [9, 10, 11, 12, 13, 14, 15]. Additionally, studies have shown that RAAS blockade utilizing angiotensin converting enzyme inhibitor(s) and angiotensin type 1 blocker(s) abrogate islet remodeling changes including fibrosis [9, 10, 11, 12, 13, 14, 15].

Angiotensin II significantly impairs pancreatic islet blood flow in isolated perfused rat pancreata and inhibits insulin release from isolated mouse islets in response to high glucose due to high proinsulin synthesis [10, 16] and both of these abnormalities are improved with RAAS blockade [10, 16]. Angiotensin II is also known to activate the NAD(P)H oxidase enzyme, which results in islet oxidative stress due to the excess production of reactive oxygen species within the islet [17, 18, 19, 20, 21]. The islets have a limited ability to handle oxidative stress making them quite vulnerable to functional and structural remodeling changes [22, 23, 24].

Insulin resistance resulting in compensatory elevated levels of insulin, proinsulin and amylin [18] may activate the local islet RAAS [25, 26] and once depleted hyperglycemia continues RAAS activation, which causes additional damage to these vulnerable islets [18, 27]. RAAS expression is up-regulated in the endocrine pancreas in T2DM and blockade with converting enzyme inhibitors and angiotensin type 1 blockers result in improvement of both islet function and structure in animal models of T2DM.

The purpose of this investigation was to examine the early ultrastructural changes of extracellular matrix remodeling fibrosis and the associated cellular remodeling of these fibrotic changes in the Ren2 model as compared to the Sprague Dawley control model. The finding of pericyte hyperplasia, activation, migration and its close association with early fibrillar-banded collagen in the islet exocrine interface was unexpected. Further, these observational findings support a role for the pericyte as a possible pancreatic stellate cell-myofibroblast precursor in this model of early peri-islet/islet exocrine interface remodeling fibrosis.

MATERIALS AND METHODS

Animals

Four male Ren2 rats (254.8 g mean body weight) and four age-matched male Sprague Dawley control rats (274.0 g mean body weight) were sacrificed at 9.3-10.2 weeks of age.

Determination of Systolic Blood Pressure and Total Body Weight

Restraint conditioning was initiated on the day of initial systolic blood pressure measurement and measured in triplicate, on separate occasions throughout the day, using the tail-cuff method (Student Oscillometric Recorder Harvard Systems Hatternas Instrument Inc., Cary, NC, USA) on the day prior to sacrifice. Total body weights were obtained at time of sacrifice.

Determination of Glucose

Following an overnight fast, blood was collected prior to sacrifice from the tail vein to determine baseline glucose. Glucose levels were determined on whole blood samples using a glucose oxidase method (OneTouch Ultra glucose analyzer; Lifescan, Inc., Milpitas, CA, USA).

Transmission Electron Microscopy (TEM)

Following harvesting, the tail sections of the pancreatic tissue was thinly sliced and placed in primary electron microscopy fixative (2% glutaraldehyde, 2 % paraformaldehyde in 0.1 M Na cacodylate buffer, pH 7.35). A Pelco 3440 Laboratory Microwave Oven (Ted Pella, Inc., Redding, CA, USA) was utilized for secondary fixation, with acetone dehydration and Epon-Spurr’s resin infiltration. Specimens were placed on a rocker overnight at room temperature and embedded the following morning and polymerized at 60°C for 24 hours. A microtome (Ultracut UCT, Leica Microsystems GmbH, Wetzlar, Germany) with a 45 degree diamond knife (Diatome, Biel, Switzerland) was used to prepare 85 nm thin sections. The specimens were then stained with 5% uranyl acetate and Sato’s triple lead stain. A transmission electron microscope (1200-EX, Jeol, Ltd., Tokyo, Japan) was utilized to view all pancreatic samples.

Light Microscopic Histological Preparation and Staining

All pancreatic tissues were harvested from the tail region of the pancreas of all rats, immediately fixed in 3% paraformaldehyde, infiltrated and embedded in paraffin.

Picrosirius Red

Picrosirius red specifically stains types I and III collagen and is birefringent when visualized by crossed polarized light. This staining procedure provides a good measure of tissue fibrosis.

Five microns sections were deparaffinized in xylene, rehydrated in ethanol series and HEPES wash buffer. The sections were stained with 0.2% phosphomolybdic acid for two minutes, rinsed in distilled water and counter-stained with picrosirius red (F3BA, saturated picric acid 400 mL, sirius red 0.4 g) for 110 minutes followed by 0.01 N hydrochloric acid treatment for 2 minutes. Then, the sections were dehydrated in reverse ethanol series, cleared, air dried, and mounted with a synthetic mounting media. The slides were observed under a microscope (Eclipse 50i, Nikon, Tokyo, Japan) and the images were captured and analyzed by MetaVue (Boyce Scientific Inc, Gray Summit, MO, USA) by using a cool camera (SNAP cf, Boyce Scientific Inc, Gray Summit, MO, USA).

Alpha Smooth Muscle Actin (alpha-SMA)

Alpha-SMA antibody staining was utilized to specifically detect activation of profibrogenic cells in tissue and has been used to detect microvascular pericytes in systemic sclerosis [28].

Tissue blocks were washed in PBS, suspended in 6.8% sucrose in PBS (pH 7.4), dehydrated in 100% acetone (60 min at 4°C), and embedded in (3 h at 4°C; no. 7022 2224- 861; Historesin Plus Leica, Deerfield, IL, USA); alternatively 1 mm3 cubes were washed in PBS, dehydrated in cold ethanol at -4 to -20°C, embedded in Unicryl® resin (British BioCell, Cardiff, UK), and polymerized by ultraviolet light (48 h at -10 to -20°C). Two-micrometer sections (Microme HM 355, Richard Allen, St. Louis, MO, USA) were stored at 4°C until use; 90- nm Unicryl® sections were picked up onto formvar-coated nickel grids and stored at room temperature until use.

3-Nitrotyrosine

Detecting the presence of 3-nitrotyrosine is an indirect method to evaluate tissue for the presence of excess reactive oxygen species due to oxidation of nitric oxide and the generation of peroxynitrite, which leads to nitration of tyrosine residues of protein in tissues. The cellular production of highly reactive nitrogen species derived from nitric oxide, such as peroxynitrite, nitrogen dioxide and nitryl chloride, leads to the nitration of tyrosine resides in tissue proteins. The extent of protein nitrotyrosine formation provides an index of the production of reactive nitrogen species and potential cell damage over a period of time.

3-nitrotyrosine content was measured as previously described [4]. Briefly, the pancreas sections were deparafinized, rehydrated, and epitopes were retrieved in citrate buffer. Endogenous peroxidases were quenched with 3% H2O2 and non-specific binding sites were blocked with avidin, biotin, and protein block (Dako, Carpinteria, CA, USA). The sections were then incubated with 1:200 primary rabbit polyclonal anti-nitrotyrosine antibody (Chemicon, Temecula, CA, USA). Sections were then washed and incubated with secondary antibodies, linked, and labeled with Strepavidin® (Dako, Carpinteria, CA, USA) for 30 min each. After several rinses with distilled water, diaminobenzidine was applied for 10 min. The sections were again rinsed with distilled water, stained with hematoxylin for 1 min, rehydrated, and mounted with a permanent media.

ETHICS

All animal procedures were approved by the University of Missouri animal care and use committees and housed/harvested in accordance with NIH guidelines.

STATISTICS

Data are reported as mean±SEM and was statistically evaluated using the Student's ttest for independent samples. Differences between experimental results were considered significant if the two-tailed P value was less than 0.05. No quantitation regarding image analysis was undertaken and therefore no statistical data is presented regarding image analysis.

RESULTS

The Peri-islet/Islet Exocrine Interface

Low power images less than 10,000 times magnification of the peri-islet region (typical of light microscopy and even low power images by TEM) appear to be quiescent in the Sprague Dawley and Ren2 animal models (Figures 1 and 2); however, on higher magnification this anatomical region seems to reflect considerable remodeling changes and important cellular activity as compared to the Sprague Dawley control model (Figure 2). There were no notable observational differences between the Ren2 and Sprague Dawley rats regarding beta-cell apoptosis, beta-cell ultrastructural organelles (mitochondria, endoplasmic reticulum, insulin secretory granules, Golgi apparatus and nuclear contents) or intra-islet fibrosis.

The peri-islet/islet exocrine interface is an important anatomical and functional region between the endocrine and exocrine pancreas, which contains the neurovascular supply including the afferent and efferent vessels (Figure 2) [29]. The islet exocrine interface region allows for communication between the islet and the exocrine pancreas and has been shown to develop significant fibrosis in the Zucker obese model of T2DM as well as T2DM in humans, which is also associated with islet amyloid deposition both within the islet and this specialized peri-islet - interface location [15, 29, 30].

The presence of fibrillar-banded collagen was eminent in the Ren2 model at the islet exocrine interface but this was not observed in the Sprague Dawley model pancreas (Figure 2). As a result of these findings it was hypothesized that there might exist a morphological change in the islet exocrine interface pericyte, which could possibly be responsible for this fibrillar-banded collagen deposition. It is known that the pericyte can differentiate into multiple cell types including myofibroblasts capable of synthesizing and depositing fibrillar-banded collagen [31, 32, 33]. Subsequently, the decision was made to examine multiple capillaries within islet exocrine interface to evaluate any changes of the pericyte that might support a role for its relationship to the fibrillar-banded collagen and the very early fibrosis observed within the islet exocrine interface.

Novel Pericyte Findings

The novel finding of pericyte proliferation, migration, hypertrophy (Figures 3, 4) was further supported by the finding of actin filaments in pericyte foot processes on higher magnification suggesting activation (Figure 5). Additionally, there was a morphological association of fibrillar-banded collagen with the activated pericyte in the islet exocrine interface of the Ren2 rats at 9-10 weeks of age (Figure 4d). Since the initial submission of this paper, our group has found direct image evidence in the human islet amyloid polypeptide rat model of type 2 diabetes mellitus (harboring the human amylin gene) of the pericyte actively extruding banded collagen fibrils from the pericyte (unpublished data). These structural changes within the extracellular matrix of the islet exocrine interface were felt to represent a very early fibrosis and these extracellular matrix and cellular remodeling changes of the pericyte were not noted in the age-matched Sprague Dawley control model. To our knowledge this is the first TEM investigation demonstrating pericyte cellular differentiation of this vascular mural cell into a fibrogenic cell such as the pancreatic stellate cellmyofibroblast in the Ren2 model of early fibrosis, hypertension and insulin resistance. There was no evidence of peri-islet/interface fibrosis in the Sprague Dawley or Ren2 model utilizing picrosirius red staining and crossed polarized light (Figure 6ab). These picrosirius red findings have also been substantiated by utilizing the Verhoeff Van Gieson staining method for fibrosis (data not shown). These findings support the notion that peri-islet/islet exocrine interface changes of fibrosis occur very early in the Ren2 model and can only be identified utilizing TEM imaging. Importantly, there was no exocrine interlobular fibrosis in the Sprague Dawley control model; however, there was a definite increase in the exocrine interlobular extracellular matrix in the Ren2 model (Figure 6c), which was felt to be indicative of exocrine interlobular fibrosis. These remodeling changes may indicate a differential spatial and temporal remodeling within the pancreas of the Ren2 rats, in that, interlobular fibrosis occurs earlier and is initially more robust than peri-islet/interface fibrosis.

Pericyte Activation

The previous observations prompted staining for early fibrotic cellular activation with alpha-smooth muscle actin (alpha-SMA). The Sprague Dawley control model rarely demonstrated signals for alpha-SMA antibody staining and when rarely identified, demonstrated only faint signals in the microvessels of the exocrine pancreas (Figure 7a). In contrast, strong signals were readily observed for alpha-SMA antibody staining in the interlobular areas of the vessels and ducts (Figure 7b) supporting the finding of interlobular fibrosis (Figure 6c). Importantly, there were strong signals present in the microvessels of the peri-islet/interface areas of the Ren2 model (Figure 7cd). These findings suggest that peri-islet microvascular mural cells, pericytes, and ductal cells were activated in the Ren2 model, which contribute to the early peri-islet/islet exocrine interface fibrillar-banded collagen as well as the exocrine interlobular fibrosis noted by TEM (Figures 2cd, 4cd, 6, 10d). We suggest that that this peri-islet/islet exocrine interface fibrosis would become more pronounced as the Ren2 model ages.

Islet Oxidative Stress

We have previously documented increased tissue organ oxidative stress in the myocardium, renal tubules and glomeruli, skeletal muscle and systemic vasculature of the Ren2 model [3, 4, 5, 6, 7, 8]. Additionally, others have found evidence of increased islet oxidative stress in the Zucker obese animal model of T2DM demonstrating that nitrotyrosine staining was strongly correlated with islet fibrosis and that this was abrogated by treatment with converting enzyme inhibitors and angiotensin type 1 blockers [15]. Recently, we have also documented this finding in a human patient with T2DM at autopsy [29, 30]. Therefore, the staining for the presence of 3-nitrotyrosine seemed imperative in this study and we were able to document a diffuse endocrine intra-islet staining for 3-nitrotyrosine in the Ren2 model, which was not present in the Sprague Dawley control model (Figure 8).

Weights, Blood Pressure and Blood Glucose

The Ren2 animals had lower mean body weights, higher blood pressures and higher blood sugars throughout the study period (Table 1). These findings were comparable to those found by previous investigators who have studied this model [3, 8, 34].

DISCUSSION

The unique finding of early fibrotic changes in the peri-islet/islet exocrine interface in this young Ren2 model of hypertension and insulin resistance [34], with known renin overexpression and most probably an increased local islet tissue RAAS (currently not proven) and increased islet oxidative stress (Figure 8), prompted further speculation.

As a result of the present findings it is currently speculated that the pericyte within the interface region is capable of differentiating into a cell type recently described and termed the pancreatic stellate cell. The pancreatic stellate cell was originally described in the pancreas almost a decade ago [35, 36] and has recently been of topical interest regarding the associated exocrine fibrosis found in various models of pancreatitis [37, 38].

As a result of our current observational findings we propose that the pool of fibrogenic pancreatic stellate cells includes not only the native fibroblasts, circulating stem cells, epithelial-acinar mesenchymal transition cells but also the differentiated microvascular pericyte. Some of the possible signaling pathways involved with the proliferation, migration, hypertrophy and activation of the pericyte-pancreatic stellate cell are presented (Figure 9).

The normal islet matrix capsule in the Sprague Dawley control model is somewhat reminiscent and morphologically similar in appearance to the glomerular basement membrane when visualized at lower TEM magnification (Figure 10ab); however, when viewed at higher magnification there is a suggestion of very faint banding at approximately 10 nanometers (Figure 10c). This matrix capsule is known to contain predominately type VI collagen as compared to types I, II or IV collagen [39, 40]. This is in contrast to the findings in the Ren2 model, in that; the Ren2 rats frequently demonstrated orderly fibrillar-banded collagen typical of very early fibrosis at the islet exocrine interface of Ren2 rat islets (Figures 2cd; 10d).

The Emerging Role of the Pericyte- Pancreatic Stellate Cell in Early Peri-Islet Fibrosis

The potential role of the pericyte as a pancreatic stellate cell-myofibroblast precursor in the Ren2 model of early periislet/ interface fibrosis merits further discussion. First, a number of studies have highlighted the in vivo capacity of the pericyte to act as mesenchymal precursor cells [34, 41, 42, 43, 44, 45, 46]. Second, during liver and renal fibrosis, resident pericyte cells have been shown to differentiate into myofibroblasts [41, 46]. The objective of our study was to investigate both early cellular and extracellular matrix remodeling and we unexpectedly found the possible relationship of the pericyte being a precursor to the pancreatic stellate cell-myofibroblast celllines capable of synthesizing fibrillar-banded collagen within the anatomical region of the peri-islet/islet exocrine interface. This evidence is now even stronger, since our group has recently observed images demonstrating islet exocrine interface pericytes actively extruding banded collagen fibrils in the human islet amyloid polypeptide rat model of type 2 diabetes mellitus (unpublished data).

The pericyte is a mesenchymal, pluripotent, ubiquitous, requisite and vascular smooth muscle-like mural cell found throughout the microvasculature (Figure 11a). It plays an important role as a regulator of stabilization, vascular development, maturation and microvessel physiological repair and pathological cellular and extracellular remodeling processes. It is located immediately adjacent to the endothelial cell where it shares physical connections referred to as peg sockets and adherens junctions (Figure 11bcd) [30, 47, 48, 49]. In addition to the cell-cell physical connections the pericyte has cell-cell molecular connections termed connexin(s) with connexin 43 being the primary connexin associated with pericyte/endothelial cell interactions. These connexins allow for the transfer of small molecules such as calcium ions, sodium ions, potassium ions and adenosine triphosphate, which are necessary for maintenance of cellular homeostasis including proliferation and differentiation [50]. The pericytes numerous foot processes-pseudopods literally embrace or envelop the endothelium (Figure 11ab) and are also capable of reaching out long distances to more distant endothelial cells or its surrounding matrix (Figures 3, 4, 11). It provides protection and physical support to the endothelium and nourishes the endothelial cell [50, 51, 52]. The pericyte has previously been referred to as the guardian angel of the endothelial cell and will literally lay down its life for the endothelial cell [51, 52]. Once this pericyte loss (due to apoptosis or necrosis) occurs in retinas of diabetic models, the endothelial cell becomes extremely vulnerable to the effects of reactive oxygen species/oxidative stress and glucotoxicity resulting in acellular capillaries and microaneurysms [50].

Recently, due to findings in genetic mouse models, failure of pericyte-endothelial cell interactions have been associated with numerous human pathological conditions including angiogenesis, diabetic microangiopathy, vascular calcification and resulted in severe or even lethal cardiovascular defects [32, 51]. Since the pericyte is pluripotent, plastic and capable of proliferation, migration, hypertrophy and activation into a fibrogenic cell (Figure 12), could the pericyte possibly be the clevis or linchpin that links microvascular damage and endothelial dysfunction to pancreatic peri-islet/islet exocrine interface and interlobular exocrine fibrosis?

In conclusion, the peri-islet/islet exocrine interface findings are preliminary, qualitative and observational; however, they are quite suggestive for supporting the role of excess local tissue islet renin, angiotensin II and oxidative stress being responsible for the very early fibrotic changes found in this young Ren2 model of hypertension and insulin resistance. Further, these observational findings support the role of the pericyte as a possible pancreatic stellate cell-myofibroblast precursor in this model of early peri-islet/islet interface remodeling fibrosis.

Hopefully, these findings will support a proof of concept and stimulate more in-depth investigations of T2DM animal models as well as T2DM islet and pancreatic tissue in humans. This model with its islet and periislet cellular and extracellular remodeling fibrosis would provide an excellent model to evaluate renin-angiotensin-aldosterone blockade and direct renin inhibition to see if they could abrogate these very early islet remodeling changes in the Ren2 model. Further utilization of light microscopy with special staining and lighting to help confirm these TEM findings and the use of different treatment modalities may elucidate the role of excess islet and pancreatic angiotensin II and the associated role of the pericyte-pancreatic stellate cell not only in the peri-islet/islet exocrine interface but also in the interlobular exocrine pancreatic regions. Additionally, we feel our observational findings may help to better understand the development of T2DM in those patients with chronic pancreatitis.

Acknowledgment

This research was supported by the investigator initiated grants NIH (R01 HL73101-01A1), the Veterans Affairs Merit System (0018) grant and Novartis Pharmaceuticals. Male transgenic Ren2 rats and male Sprague-Dawley controls were kindly provided by Dr. Carlos M. Ferrario, Wake Forest University School of Medicine, Winston-Salem, North Carolina through the Transgenic Core Facility supported in part by NIH grant HL-51952. The authors would like to acknowledge the Electron Microscopic Core Center at the University of Missouri, Columbia, Missouri for their excellent help and tissue preparation of animal samples for viewing.

Conflict of interest

The authors have no potential conflicts of interest

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